We have previously demonstrated that rat and human liver HMG-CoA reductase activity is modulated in vitro and in vivo in a bicyclic cascade system involving reversible phosphorylation of both HMG-CoA reductase and reductase kinase. Recently, we have also reported the modulation of the enzymic activity of both soluble purified (Mr 53,000) and native (Mr 100,000) HMG-CoA reductase involving a Ca2+-activated and phospholipid-dependent protein kinase C-mediated phosphorylation. During the past year we have purified and characterized a low molecular weight Ca2+, calmodulin-dependent protein kinase (Mr 110,000) from rat brain cytosol. This purified protein kinase is different from other known calmodulin-dependent kinases (Mr 500-600,000). The new kinase also differs in terms of its degree of autophosphorylation and specificity toward other substrates including HMG-CoA reductase. Maximal phosphorylation of purified HMG-CoA reductase was approximately one mol/mol of 100,000 native HMG-CoA reductase. Dephosphorylation of 32P-HMG-CoA reductase was associated with complete reactivation of HMG-CoA reductase activity and near total loss of radioactivity. Ca2+ calmodulin-dependent kinase is able to phosphorylate two different sites in the purified HMG-CoA reductase molecule. Phosphoaminoacid analysis of each phosphopeptide revealed that only serine residues are phosphorylated by calmodulin-dependent kinase. Based on these results and our previous in vitro and in vivo studies, we now propose that both native and purified HMG-CoA reductase activity is modulated by reversible covalent phosphorylation involving three separate kinase systems including reductase kinase, protein kinase C, and Ca2+, calmodulin-dependent protein kinase.